Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration. METHODS: To define the functional role of CXCL12 in the progression of glomerular disease, we used the CXCL12-specific inhibitor NOX-A12, an L: -enantiomeric RNA oligonucleotide (Spiegelmer). A mouse model of type 2 diabetes (db/db mice) was used. Male db/db mice, uni-nephrectomised at 6 weeks of age, received subcutaneous injections with a PEGylated form of NOX-A12, non-functional control Spiegelmer or vehicle on alternate days from 4 to 6 months of age. RESULTS: Immunostaining localised renal CXCL12 production to glomerular podocytes in db/db mice with early or advanced diabetic nephropathy. CXCL12 inhibition significantly reduced the degree of glomerulosclerosis, increased the number of podocytes, prevented the onset of albuminuria and maintained the peritubular vasculature without affecting blood glucose levels, body weight or glomerular macrophage infiltration. CONCLUSIONS/INTERPRETATION: We conclude that podocytes produce CXCL12, which contributes to proteinuria and glomerulosclerosis in our mouse model of type 2 diabetes. This novel pathomechanism provides the first evidence that CXCL12 could be a therapeutic target in (diabetic) glomerulosclerosis.

Comparison of alanine production after L-leucine and AMP deamination in an enzymatic model and in muscle specimens.

The amination of 2-oxoglutarate to glutamate by the deamination of leucine followed by the transamination of pyruvate to alanine in skeletal muscle is generally accepted. However, alanine formation following AMP deamination by AMP deaminase is still questionable even though it is theoretically possible. For this reason, we investigated in an enzymatic model the dependence of alanine yield both on the increasing concentration of AMP and leucine as amino group donors as well as on AMP deaminase and leucine dehydrogenase augmentation. Up to a concentration of 375 microM neither of the amino group donors produced a difference in the glutamate nor alanine yield. At a concentration of 500 microM ammonia formation was less, but alanine production was higher when leucine was present as a starting material. However, in muscle samples obtained from trained or untrained rats we did not find an increase in alanine yield when AMP was added to the muscle sample, even though NH3 production was significantly higher in samples with than in those without AMP. This discrepancy might be further elucidated by hindquarter perfusion experiments, in which alanine release would be determined after AMP deamination enhanced by electrostimulation of these muscle groups.

Electrochemical detection of catecholamines in urine and plasma after separation with HPLC.

The catecholamines in plasma and urine were determined by electrochemical detection after separation on a HPLC column RCM 100 C 18. Linear calibration plots of epinephrine, norepinephrine and dopamine have been obtained in the range expected to appear in urine or plasma. Coefficients of variation (0.5 to 10%), recovery rates (60 to 80%) and the standard deviation were satisfactory, whereas plasma dopamine showed a greater variation (10-15%). The values ascertained by this technique were compared with those determined by the radioenzymatic assay of Da Prada et al. They showed a good correlation for norepinephrine r = 0.92, p less than 0.001 and epinephrine r = 0.80, p less than 0.01, but less for dopamine r = 0.1. Experimental details of isolation from plasma with Al2O3 adsorption and desorption with HClO4, and from urine with ion exchange chromatography on Bio-Rex 70 and elution with boric acid are described. Technical modifications which improve the method are reported. The optimized assay could reliably be employed in investigations of more than 3000 urine and plasma samples obtained from patients and athletes before and after exercise.

Determination of alanine, lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate by flow injection analysis (FIA).

The flow infection analysis (FIA) described by Ruzicka and Hansen was adjusted for lactic acid determination by Rydevik et al. We were able to elaborate some other NAD or NADH-dependent enzyme reactions with the FIA system. The reliability of the alanine assay corresponds to that of the lactate FIA method. The coefficient of variation was on an average 2.8%; th sample rate was 60/h with consistent duplicates. The beta-OHB assay had almost the same validity. Pyruvate, however, was less reliable with higher coefficients of variation. ACAC FIA assay, which was suitable for the determination in acid solution after neutralization, could not yet be employed for the determination in serum. In comparison to the manual enzyme methods, the FIA assays described here indicated a higher sampling rate, a lower reagent consumption, a lower coefficient of variation, a better reproducibility, and a greater consistency of duplicates.

Influence of training and anabolic steroids on the LDH isozyme pattern of skeletal and heart muscle fibers of guinea pigs.

The influence of training and anabolic steroids (methandrostenolone) on the enzyme activity of the LDH system was investigated in 72 male guinea pigs. Sedentary animals and animals subjected to two different training regimens with and without anabolic steroids were compared in six groups each consisting of 12 guinea pigs. The training was performed on a rodent treadmill for 1 month, 30 min/day at an inclination of 45 degrees or 5 degrees, and at a speed of 30 m/min. Total LDH, its isozymes, and the composition of M and H subunits were determined enzymatically and by electrophoretical separation on cellulose acetate membrane in the following muscle fiber types; m. vastus lateralis white (FG) and red portion (FOG), m. soleus (SO), m. psoas major white (FG) and red portion (FOG), and in the left ventricle of the heart. The administration of anabolic steroids did not change the activity of total LDH or the composition of M and H subunits in sedentary animals. Exercise at 5 degrees inclination significantly reduced the total LDH and the concentration of H subunits in FOG, SO, and heart muscle fibers but not in FG muscle fibers. The additional application of anabolic steroids intensified these changes and also reduced the total LDH and the H subunit in FG as well as the M subunit in FOG, SO, and heart muscle fibers. After exercise at 45 degrees inclination with and without the application of anabolic steroids, the activity of LDH and its isozymes was identical to the values obtained in untrained animals but significantly higher than after the training at 5 degrees inclination. The typing of FG, FOG, and SO muscle fibers by the LDH isozyme pattern was not satisfactory, especially for the discrimination between FOG and SO fibers but also between FG and FOG fibers of muscle with different anatomic functions both in sedentary and trained animals.

[The use of an EDP time sharing system]. / Die Nutzung eines EDV-Teilnehmersystems

Mathematical problems in pharmacokinetics can be solved in a simple and effective way by using a computer timesharing system. It is reasonable to make use of such a system because the capabilities of a large scale computer are available without the prerequisite of special knowledge in data processing. Besides the possibilities of individual and simple programming, there are predefined programs available to solve special problems: mathematical statistics, plotting of time series etc.